This invention is directed to the synthesis and use of oligonucleotides for eliciting RNase H activity for strand cleavage in an opposing strand. Included in the invention are oligonucleotides wherein at least some of the nucleoside units of the oligonucleotides are functionalized to be nuclease resistant, at least some of the nucleoside units of the oligonucleotides include a substituent that potentiates hybridization of the oligonucleotide to a complementary strand of nucleic acid, and at least some of the nucleoside units of the oligonucleotides include 2xe2x80x2-deoxy-erythro-pentofuranosyl sugar moieties. The oligonucleotides are useful for therapeutics, diagnostics and as research reagents.
Oligonucleotides are known to hybridize to single-stranded DNA or RNA molecules. Hybridization is the sequence-specific base pair hydrogen bonding of nucleobases of the oligonucleotides to nucleobases of target DNA or RNA. Such nucleobase pairs are said to be complementary to one another.
In determining the extent of hybridization of an oligonucleotide to a complementary nucleic acid, the relative ability of an oligonucleotide to bind to the complementary nucleic acid may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (Tm), a characteristic physical property of double helices, denotes the temperature (in degrees centigrade) at which 50% helical (hybridized) versus coil (unhybridized) forms are present. Tm is measured by using the UV spectrum to determine the formation and breakdown (melting) of the hybridization complex. Base stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the bonds between the strands.
Oligonucleotides can be used to effect enzymatic cleavage of a target RNA by using the intracellular enzyme, RNase H. The mechanism of such RNase H cleavage is believed to require that a 2xe2x80x2-deoxyribofuranosyl oligonucleotide hybridize to a target RNA. The resulting DNA-RNA duplex activates the RNase H enzyme and the activated enzyme cleaves the RNA strand. Cleavage of the RNA strand destroys the normal function of the RNA. Phosphorothioate oligonucleotides are believed to operate via this type of mechanism. However, for a DNA oligonucleotide to be useful for cellular activation of RNase H, the oligonucleotide preferably is reasonably stable to nucleases in order to survive in cells for a time period sufficient for RNase H activation. For non-cellular uses, such as use of oligonucleotides as research reagents, such nuclease stability may not be necessary.
Several publications describe the interaction of RNase H and oligonucleotides. Of particular interest are: (1) Dagle et al., Nucleic Acids Research, 1990, 18, 4751; (2) Dagle et al., Antisense Research And Development, 1991, 1, 11; (3) Eder et al., J. Biol. Chem., 1991, 266, 6472; and (4) Dagle et al., Nucleic Acids Research, 1991, 19, 1805. According to these publications, DNA oligonucleotides having both unmodified phosphodiester internucleoside linkages and modified phosphorothioate internucleoside linkages are substrates for cellular RNase H. Since they are substrates, they activate the cleavage of target RNA by RNase H. However, the authors further noted that in Xenopus embryos, both phosphodiester linkages and phosphorothioate linkages are also subject to exonuclease degradation. Such nuclease degradation is detrimental since it rapidly depletes the oligonucleotide available for RNase H activation.
As described in references (1), (2) and (4), to stabilize oligonucleotides against nuclease degradation while still providing for RNase H activation, 2xe2x80x2-deoxy oligonucleotides having a short section of phosphodiester linked nucleosides positioned between sections of phosphoramidate, alkyl phosphonate or phosphotriester linkages were constructed. While the phosphoramidate-containing oligonucleotides were stabilized against exonucleases, in reference (4) the authors noted that each phosphoramidate linkage resulted in a loss of 1.6xc2x0 C. in the measured Tm value of the phosphoramidate-containing oligonucleotides. Such a decrease in the Tm value is indicative of a decrease in hybridization between the oligonucleotide and its target strand.
Other authors have commented on the effect such a loss of hybridization between an oligonucleotide and its target strand can have. Saison-Behmoaras et al. (EMBO Journal, 1991, 10, 1111) observed that even though an oligonucleotide could be a substrate for RNase H, cleavage efficiency by RNase H was low because of weak hybridization to the mRNA. The authors also noted that the inclusion of an acridine substitution at the 3xe2x80x2 end of the oligonucleotide protected the oligonucleotide from exonucleases.
U.S. Pat. No. 5,013,830, issued May 7, 1991, discloses mixed oligomers comprising an RNA oligomer, or a derivative thereof, conjugated to a DNA oligomer via a phosphodiester linkage. The RNA oligomers also bear 2xe2x80x2-O-alkyl substituents. However, being phosphodiesters, the oligomers are susceptible to nuclease cleavage.
European Patent application 339,842, filed Apr. 13, 1989, discloses 2xe2x80x2-O-substituted phosphorothioate oligonucleotides, including 2xe2x80x2-O-methylribooligonucleotide phosphorothioate derivatives. The above-mentioned application also discloses 2xe2x80x2-O-methyl phosphodiester oligonucleotides which lack nuclease resistance.
U.S. Pat. No. 5,149,797, issued Sep. 22, 1992, discloses mixed phosphate backbone oligonucleotides which include an internal portion of deoxynucleotides linked by phosphodiester linkages, and flanked on each side by a portion of modified DNA or RNA sequences. The flanking sequences include methyl phosphonate, phosphoromorpholidate, phosphoropiperazidate or phosphoramidate linkages.
U.S. Pat. No. 5,256,775, issued Oct. 26, 1993, describes mixed oligonucleotides that incorporate phosphoramidate linkages and phosphorothioate or phosphorodithioate linkages.
While it has been recognized that cleavage of a target RNA strand using an oligonucleotide and RNase H would be useful, nuclease resistance of the oligonucleotide and fidelity of hybridization are of great importance in the development of oligonucleotide therapeutics. Accordingly, there remains a long-felt need for methods and materials that could activate RNase H while concurrently maintaining or improving hybridization properties and providing nuclease resistance. Such oligonucleotides are also desired as research reagents and diagnostic agents.
In accordance with one embodiment of this invention there are provided oligonucleotides formed from a sequence of nucleoside units. The oligonucleotides incorporate a least one nucleoside unit that is functionalized to increase nuclease resistance of the oligonucleotides. Further, at least some of the nucleoside units of the oligonucleotides are functionalized with a substituent group to increase binding affinity of the oligonucleotides for target RNAs, and at least some of the nucleoside units have 2xe2x80x2-deoxy-erythro-pentofuranosyl sugar moieties.
In preferred oligonucleotides of the present invention, nucleoside units which are functionalized for increasing binding affinity include a 2xe2x80x2-substituent group. In preferred embodiments, the 2xe2x80x2-substituent group includes fluoro, C1-C20 alkoxy, C1-C9 aminoalkoxy, including aminopropoxy, allyloxy, imidazolylalkoxy and polyethylene glycol. Preferred alkoxy substituents include methoxy, ethoxy and propoxy. A preferred aminoalkoxy unit is aminopropoxy. A preferred imidazolylalkoxy substituent is imidazolylpropoxy. A preferred polyethylene glycol substituent is xe2x80x94O-ethyl-O-methyl or methoxyethoxy (xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH3).
The oligonucleotides of the present invention include nucleoside units connected by charged phosphorus linkages selected from a group consisting of phosphodiester and phosphorothioate linkages.
The oligonucleotides of the present invention include a plurality of linked nucleoside units bearing substituent groups that increase binding affinity of the oligonucleotide to a complementary strand of nucleic acid. In certain preferred embodiments, the sequence of an oligonucleotide having nucleoside units that bear such substituents can be divided into a first subsequence and a second subsequence, with the first subsequence having linked nucleoside units bearing 2xe2x80x2-substituted-erythro-pentofuranosyl sugar moieties and the second subsequence having linked nucleoside units bearing 2xe2x80x2-deoxy-erythro-pentofuranosyl sugar moieties. Preferably, said second subsequence has at least three nucleoside units, and more preferably, has at least five nucleoside units. In further preferred embodiments there exists a third subsequence, the nucleoside units of which are selected from those which are selectable for the first subsequence. It is preferred that the second subsequence be positioned between the first and the third subsequences. Such oligonucleotides of the present invention are also referred to as xe2x80x9cchimeras,xe2x80x9d or xe2x80x9cchimericxe2x80x9d or xe2x80x9cgappedxe2x80x9d oligonucleotides.
In further preferred oligonucleotides of the invention, nucleoside units bearing substituents that increase binding affinity are located at one or both of the 3xe2x80x2 or the 5xe2x80x2 termini of the oligonucleotide. There can be from one to about eight nucleoside units that are substituted with substituent groups. Preferably, at least five nucleoside units bear 2xe2x80x2-deoxy-erythro-pentofuranosyl sugar moieties.
The nucleoside units of the oligonucleotides of the present invention comprise nucleobases linked to 2xe2x80x2-substituted and 2xe2x80x2-deoxy-erythro-pentofuranosyl sugar moieties by phosphorus linkages such as phosphodiester and phosphorothioate linkages. Preferred nucleobases of the invention include purines and pyrimidines such as adenine, guanine, cytosine, uridine, and thymine, as well as other synthetic and natural nucleobases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, and 7-methylguanine. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley and Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.
The invention also provides methods for treating an organism having a disease characterized by the undesired production of a protein. These methods include contacting the organism with an oligonucleotide having a sequence of nucleoside units capable of specifically hybridizing with a complementary strand of nucleic acid with at least one of the nucleoside units being functionalized to increase nuclease resistance of the oligonucleotide to nucleases, with a substituent group located thereon to increase binding affinity of the oligonucleotide to the complementary strand of nucleic acid, and with a plurality of the nucleoside units having 2xe2x80x2-deoxy-erythro-pentofuranosyl sugar moieties.
Further in accordance with the present invention there are provided compositions including a pharmaceutically effective amount of an oligonucleotide having a sequence of nucleoside units capable of specifically hybridizing with a complementary strand of nucleic acid having at least one of the nucleoside units functionalized to increase nuclease resistance of the oligonucleotide to nucleases, wherein a plurality of the nucleoside units have a substituent group located thereon to increase binding affinity of the oligonucleotide to the complementary strand of nucleic acid, and wherein a plurality of the nucleoside units have 2xe2x80x2-deoxy-erythro-pentofuranosyl sugar moieties. The compositions further include a pharmaceutically acceptable diluent or carrier.
Further in accordance with this invention there are provided methods for in vitro modification of a sequence-specific nucleic acid including contacting a test solution containing an RNase H enzyme and said nucleic acid with an oligonucleotide having a sequence of nucleoside units capable of specifically hybridizing to a complementary strand of the nucleic acid, where at least one of the nucleoside units is functionalized to increase nuclease resistance of the oligonucleotide to nucleases, where a plurality of the nucleoside units have a substituent group located thereon to increase binding affinity of the oligonucleotide to the complementary strand of nucleic acid, and where a plurality of the nucleoside units have 2xe2x80x2-deoxy-erythro-pentofuranosyl sugar moieties.
There are also provided methods of concurrently enhancing hybridization and RNase H enzyme activation in an organism that includes contacting the organism with an oligonucleotide having a sequence of nucleoside units capable of specifically hybridizing to a complementary strand of nucleic acid, where at least one of the nucleoside units is functionalized to increase nuclease resistance of the oligonucleotide to nucleases, where a plurality of the nucleoside units have a substituent group located thereon to increase binding affinity of the oligonucleotide to the complementary strand of nucleic acid, and where a plurality of the nucleoside units have 2xe2x80x2-deoxy-erythro-pentofuranosyl sugar moieties.
The invention further provides diagnostic methods for detecting the presence or absence of abnormal RNA molecules, or abnormal or inappropriate expression of normal RNA molecules in organisms or cells.